TRB3 (also known as Nerve Growth Factor-induced Protein Kinase (NIPK); p65-interacting inhibitor of NFκB (SINK); or SKIP3) was first identified as a neuronal cell death-inducible putative protein kinase (NIPK) in the rat. TRB3 and its related family members TRB1 and TRB2 share 45% sequence identity overall and bear strong resemblances to Tribbles, a Drosophila protein that inhibits mitosis early in development by binding to the CDC25 homolog string and promoting its ubiquitination and proteasome-mediated degradation (Grosshans and Wieschaus, Cell, 101:523-31, 2000; Mata et al., Cell, 101:511-22, 2000; Rorth et al., Mol. Cell., 6:23-30, 2000; Seher and Leptin, Curr. Biol., 10:623-9, 2000). Like Tribbles, TRB family members have a truncated kinase domain that lacks an adenosine 5′-triphosphate binding site (GXGXXG) and contains a variant catalytic core motif within the Ser/Thr kinase domain (e.g., LRDLKLRR in mouse TRB3 (residues 180-187 of SEQ ID NO: 2) as compared to the catalytic core consensus sequence HRDLKPEN; SEQ ID NO: 21). Correspondingly, Tribbles and its mammalian counterparts lack detectable kinase activity by in vitro kinase assay, and are thought to have evolved instead as adaptor proteins (Du et al., Science, 300:1574-1577, 2003).
Using a yeast 2-hybrid assay, Du et al. identified TRB3 as a protein from a preadipocyte cDNA library that interacted with AKT1 (Science, 300:1574-1577, 2003). TRB3 disrupted insulin signaling in liver by binding directly to AKT and inhibiting activation of the kinase (Du et al., Science, 300:1574-1577, 2003). Under fasting conditions, TRB3 expression was induced in liver. Amounts of TRB3 RNA and protein also were increased in livers of db/db diabetic mice compared with those of wild-type mice, and hepatic overexpression of TRB3 in amounts comparable to those in db/db mice promoted hyperglycemia and glucose intolerance. Du et al. (Science, 300:1574-1577, 2003) suggested that by interfering with AKT activation, TRB3 contributed to insulin resistance in individuals with susceptibility to type 2 diabetes. Indeed, humans with a gain of function mutation in TRB3 have a higher incidence of insulin resistance and diabetes-associated complications (Prudente et al., Diabetes, 54:2807-11, 2005).
Wu et al. cloned TRB3, which was designated SINK, from a human B-cell cDNA library (J. Biol. Chem., 278:27072-27079, 2003). Northern blot analysis detected TRB3 mRNA in spleen, thymus, prostate, liver, and pancreas, but not in other tissues examined. Wu et al. found that overexpression of TRB3 in human embryonic kidney cells inhibited NFκB-dependent transcription induced by TNF or its downstream signaling proteins, but TRB3 did not inhibit NFκB translocation to the nucleus or binding of NFκB to DNA (J. Biol. Chem., 278:27072-27079, 2003). Coimmunoprecipitation and in vitro kinase assays indicated that TRB3 specifically interacted with the NFκB transactivator p65 and inhibited p65 phosphorylation. Consistent with its role in inhibiting NFκB-dependent transcription, TRB3 also sensitized cells to apoptosis induced by TNF and TRAIL. Wu et al. concluded that TRB3 (SINK) was involved in a negative feedback control pathway of NFκB-induced gene expression (J. Biol. Chem., 278:27072-27079, 2003).
Kiss-Toth et al. detected TRB3 expression in pancreas, peripheral blood leukocytes, and bone marrow, and found that overexpression of TRB3 in HeLa cells inhibited AP1 activity and blocked oncogenic Ras-driven AP1 activation (J. Biol. Chem., 279:42703-42708, 2004). ERK activation was enhanced by TRB3, but only at low TRB3 doses. Coimmunoprecipitation and yeast 2-hybrid assays showed that MEK1 (MAP2K1) interacted with both TRB1 and TRB3, and MKK7 (MAP2K7) interacted specifically with TRB3. Cotransfection of MKK7 enhanced the level of TRB3, indicating that the TRIB-MAPKK interaction stabilized TRB3.
TRB3 and its related family members seem to be involved in important molecular, cellular and physiological processes; although, much remains to be discovered about these interesting psuedokinases and their uses, for example, in medicine, diagnostics, and/or the discovery of prospective and/or actual therapeutics. With particular regard to diseases of metabolic function (e.g., diabetes and obesity), new methods are needed for identifying useful candidate agents and therapeutics for the prevention and/or treatment of these increasingly common and serious disorders.